PROJECT SUMMARY The cullin-ring ligase (CRL) family of E3 ubiquitin ligases play a critical role in the control of basic development, and some CRLs have been implicated in regulation of early neurodevelopmental processes. However, Cullin-9 (CUL9), has proven to be a unique member with an elusive function. CRLs generally form large complexes that ubiquitinate a set of specific substrates. CUL9 has not been shown to form large complexes and has only two identified substrates. Our data provide strong evidence that CUL9 may regulate transcription factors key for neural stem cell fate in the early neural induction phase of cortical differentiation. I have shown that CUL9 protein levels drastically increase during the neural induction phase of cortical glutamatergic differentiation. I suspect that high levels of CUL9 expression may be required for the differentiation of proliferative, self-renewing human neural precursor cells (hNPCs) with multipotent differentiation potential. CUL9 knockout mice sporadically develop enlarged brains (megalencephaly) and display autism-like behavior (unpublished observations). Megalencephaly is often the result of improper maintenance of the NPC pool in the developing cerebral cortex leading to abnormal corticogenesis. Regulation of NPC transcription factor levels, such as SOX2 and PAX6, is critical in an NPC?s decision to self-renew or differentiate. My preliminary data and the current literature provide a strong premise for my hypothesis that proper regulation of CUL9 and its substrates is required for the differentiation of self-renewing and multipotent hNPCs from human pluripotent stem cells (hPSCs). In Aim 1, I will define the role of CUL9 in maintaining self-renewal and multipotency of hNPCs. My preliminary studies demonstrate that CUL9 depleted hPSCs form enlarged embryoid bodies and fewer neural rosettes with an expanded lumen. The ability of CUL9 depleted cells to form self-renewing, multipotent hNPCs will be assessed. Additionally, the number, lumen size, and organization of neural rosettes formed from CUL9 depleted cells will be quantified. In Aim 2, I will clarify the molecular mechanism underlying CUL9 control of hNPC differentiation. My preliminary data demonstrate that hNPCs with depleted CUL9 levels express elevated levels of SOX2 and PAX6 protein. I will verify if SOX2 and/or PAX6 are CUL9 substrates by in vitro ubiquitination assays. Then, the residues targeted by CUL9 will be identified. I have also demonstrated that CUL9 and the Anaphase Promoting Complex/Cyclosome (APC/C) interact, and that depletion of the APC/C substrate adaptor protein CDC20 Homolog 1 (CDH1) results in increased CUL9 protein levels. Thus, I will characterize the CUL9-APC/C interaction and determine if CUL9 is a substrate of APC/C-CDH1. A CUL9 mutant construct unable to interact with the APC/C will be identified and the effect of its expression on neural rosette formation, and SOX2 and PAX6 protein levels and transcriptional activity will be determined. The results of these studies may reveal a novel CUL9-APC/C signaling axis required for the proper differentiation of hNPCs; my studies could also shed light into potential mechanisms that when disrupted can lead to megalencephaly and Autism Spectrum Disorder.